Isocombretastatins A: 1,1-Diarylethenes as potent inhibitors of tubulin polymerization and cytotoxic compounds

Article abstract of DOI:10.1016/j.bmc.2009.07.012

Isocombretastatins A are 1,1-diarylethene isomers of combretastatins A. We have synthesized the isomers of combretastatin A-4, deoxycombretastatin A-4, 3-amino-deoxycombretastatin A-4 (AVE-8063), naphthylcombretastatin and the N-methyl- and N-ethyl-5-indo

Full text of DOI:10.1016/j.bmc.2009.07.012

Bioorganic & Medicinal Chemistry 17 (2009) 6422–6431
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Isocombretastatins A: 1,1-Diarylethenes as potent inhibitors of tubulin
polymerization and cytotoxic compounds
Raquel Álvarez a, Concepción Álvarez a, Faustino Mollinedo b, Beatriz G. Sierra b,c, Manuel Medarde a,
,
*
Rafael Peláez a,
*
a Departamento de Química Farmacéutica, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno s/n, Salamanca E-37007, Spain
b Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain
c APOINTECH, Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Campus de Villamayor–Parque Científico, C/Río Duero 12, E-37185
Villamayor, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Isocombretastatins A are 1,1-diarylethene isomers of combretastatins A. We have synthesized the iso-
mers of combretastatin A-4, deoxycombretastatin A-4, 3-amino-deoxycombretastatin A-4 (AVE-8063),
naphthylcombretastatin and the N-methyl- and N-ethyl-5-indolyl analogues of combretastatin A-4. Ana-
logues with a 2,3,4-trimethoxyphenyl ring instead of the 3,4,5-trimethoxyphenyl ring have also been pre-
pared. The isocombretastatins A strongly inhibit tubulin polymerization and are potent cytotoxic
compounds, some of them with IC50s in the nanomolar range. This new family of tubulin inhibitors shows
higher or comparable potency when compared to phenstatin or combretastatin analogues. These results
suggest that one carbon bridges with a geminal diaryl substitution can successfully replace the two car-
bon bridge of combretastatins and that the carbonyl group of phenstatins is not essential for high
potency.
Received 1 June 2009
Revised 29 June 2009
Accepted 5 July 2009
Available online 9 July 2009
Keywords:
Tubulin
Combretastatins
Phenstatins
Colchicine
Ó 2009 Elsevier Ltd. All rights reserved.
Antitumour
Cytotoxicity
Antimitotic
1. Introduction
the colchicine site of tubulin.6 The most potent combretastatin
analogues are substituted cis-stilbenes (Fig. 1), such as combretast-
The eukaryotic microtubule system is composed of
a
-tubulin
atin A-1 and combretastatin A-4, whose phosphate prodrugs are
currently in clinical development.7 They have attracted much
attention owing to their potent cytotoxic and vascular disrupting
activities.8 The cisoid disposition of the two aromatic rings is
essential for the activity, and many attempts to prevent the easy
isomerization from cis to trans olefins have been carried out,
including the formation of small heterocycles or carbocycles on
the bridge,7 reduction of the number of atoms of the bridge,9 or for-
mation of macrocycles by linkage of the two aromatic rings.10
The phenstatins (Fig. 1) are bisarylketones which display high
potency as tubulin polymerization inhibitors.9 The phenstatins
share many of the SARs of combretastatins, such as the importance
of a trimethoxyphenyl ring (A) and the need for a non coplanar ci-
soid disposition of the two aromatic rings. The key structural ele-
ments for high activities of inhibitors of tubulin polymerization
binding at the colchicine site have been analyzed, and, recently, a
common pharmacophore has been proposed based on the consis-
tent structural features and binding interactions found in docking
models of structurally diverse colchicine site ligands.5b,c According
to this pharmacophore, an explanation for the high potency of
phenstatins has been put forward: the carbonyl oxygen would
hydrogen bond to the backbone N–H group of residues
and b-tubulin heterodimers arranged head to tail. Microtubules
and their dynamics are involved in many essential cell functions,
such as development and maintenance of shape, motility and
intracellular transport, cell signaling and cell division.1 Perturba-
tion of tubulin polymerization equilibria can produce metaphase
arrest, making microtubules attractive molecular targets for anti-
cancer therapeutics.2 Most of the antimitotic drugs in clinical use
or in clinical development bind at three major binding sites on
tubulin: the vinca, taxane, and colchicine sites.3 The colchicine site
at tubulin is very hydrophobic, as shown by the X-ray crystal struc-
tures of its complexes with DAMA-colchicine and podophyllo-
toxin.4 Many natural and synthetic compounds of diverse origin
and structure bind to the colchicine site and modify the polymer-
ization of the protein, and several models have attempted to ex-
plain the structure–activity relationships of colchicine site
ligands.5
Combretastatins are a family of compounds of natural origin
which strongly inhibit tubulin polymerization through binding to
* Corresponding authors. Tel.: +34 23 294528; fax: +34 23 294500 (R.P.).
E-mail address: pelaez@usal.es (R. Peláez).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.012

R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
6423
Figure 1. Structures of colchicine, combretastatins and phenstatins.
b248-b249-b250, thus providing one additional hydrogen bond
acceptor site (point A3) in phenstatins. Results obtained by the
modification of the carbonyl of phenstatins are also in agreement
with this model.11
Following our research on tubulin polymerization inhibitors
based on combretastatins and phenstatins, we have synthesized
a new family of substituted 1,1-diarylethenes, which we have
called isocombretastatins A12 because they are structural isomers
of well known, highly potent combretastatins A (Z-1,2-diaryleth-
enes). In order to fairly compare isocombretastatins A with com-
pharmacophoric model is not essential for high potency. Related
substituted 1,1-diarylethenes have been previously shown toinhibit
tubulin polymerization (Fig. 2), such as some alkylidenediarylme-
thanes (ADAMs) originally designed as HIV-RT inhibitors13 and
CC-5079,14 and recently the first isocombretastatins
A have
appeared.15 The series synthesized and assayed in this research
enlarge the SAR of colchicines site ligands and expand the scope of
isocombretastatins as potential tubulin polymerization inhibitors.
The 1,1-diarylmethanes were synthesized by Wittig reactions of
methylenetriphenylphosphorane with appropriately substituted
diarylketones. In turn, the phenones were obtained from diaryl-
methanols by PDC or KMnO4 oxidation, following literature proce-
dures (Scheme 1).11
The diarylmethanols were synthesized by reacting the aldehyde
of one aromatic moiety with the organolithium or organomagne-
sium aromatic salts of the other. The organometallic and aromatic
aldehyde combination used varied depending on the nature and
reactivity of the aromatic systems implied. Thus, for the synthesis
of compounds with two phenyl rings, one being 2,3,4-trimethoxy-
phenyl ring, the ortho chelating capability of the methoxy groups
was used to generate the ortho lithiated species by treatment of
1,2,3-trimethoxybenzene with n-butyllithium. On the other hand,
the diphenylmethanols with a 3,4,5-trimethoxyphenyl ring were
synthesized by reaction of the benzenic aldehydes with the mag-
nesium salt of 3,4,5-trimethoxyphenylbromide, in an attempt to
minimize transmetallation reactions to the ortho metallated posi-
tions (Scheme 2).
bretastatins
A and phenstatins, we have synthesized and
evaluated a series of isocombretastatins with aryl rings that render
the combretastatins A and phenstatins more potent.7,9,11 Thus,
compounds with a 3,4,5-trimethoxyphenyl ring combined with
either a 3-hydroxy-4-methoxyphenyl ring (CA-4 analogue), a 4-
methoxyphenyl ring (deoxyCA-4 analogue),
a
3-amino-4-
methoxyphenyl ring (AVE-8063 analogue), a 2-naphthyl moiety
(naphthylcombretastatin analogue) or a 1-methyl- or 1-ethyl-5-
indolyl system have been synthesized, assayed and compared to
their combretastatin and phenstatin analogues. Moreover, consid-
ering that the lack of the carbonyl oxygen on the bridge of phenst-
atins (considered to be an important pharmacophoric point by
establishing hydrogen bonds) could somewhat release the anchor
of the structure at that point and allow for a certain variation in
the binding mode, the analogues with a 2,3,4-trimethoxyphenyl
ring instead of the 3,4,5-trimethoxyphenyl ring have also been
synthesized and evaluated. These later analogues would mimic
the colchicine binding mode.
We have found that isocombretastatins display tubulin poly-
merization inhibitory potencies equal to or better than those of
the parent combretastatins. They also have the advantage of not
suffering from their cis–trans isomerization problems and, in terms
of structure–activity relationships, they show that the optimal
bridge length is not necessarily two atoms, and that the hydrogen
bond acceptor capacity represented by the A3 site of the
Compounds with a naphthalene ring were synthesized by reac-
tion of the naphthyllithium with appropriate aldehydes. Finally,
compounds with a N-alkyl-5-indolyl moiety were synthesized
from the 2,5-dilithium salt, in an effort to prevent transmetallation
reactions and to force reaction from the 5-indole position. Func-
tional group modifications at the ketone or olefin states, including
phenol protection/deprotection steps and nitro group reductions,
led to the desired substitutions on the phenyl rings.
N
N
O
CN
MeO
MeO
X
OMe
MeO
OMe
OMe
B
A
OMe
N
O
MeO
NC
MeO
ADAMs
CC-5079
1,1-diarylethenes
Figure 2. Structures of representative alkylidenediarylmethane inhibitors of tubulin polymerization (ADAMs and CC-5079) and the isocombretastatins A (1,1-diarylethenes).

6424
R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
Scheme 1. Synthesis of diarylketones 4a–p and isocombretastatins
methyltriphenylphosponium iodide, THF, ꢀ40 °C, nBuLi, then ketone; (iv) 4: Fe, EtOH:AcOH:H2O, HCl or 5: Zn, HCl, CH2Cl2:AcOH; (v) TBAF, THF.
A
5a–p. Reagents and conditions: (i) KMnO4, HSO4ꢀ ꢁ Bu4Nþ
, CH2Cl2; (ii) PDC, CH2Cl2; (iii)
The synthesized compounds were tested at different concentra-
tions in the tubulin polymerization assay and the IC50s were deter-
mined as previously described.11 Our results are shown in Table I,
altogether with literature values, with whom they are in good
agreement.
the trimethoxyphenyl ring in different dispositions than those
found for the reference ligands (podophyllotoxin, colchicine, CA-
4, and other colchicine site ligands similarly docked, which place
their trimethoxyphenyl rings close to those observed in the X-ray
structures). Considering the essential role of such a ring in many
diverse colchicine—site binding ligands,7 we decided to give pref-
erence to the poses docked at the podophyllotoxin site with a tri-
methoxyphenyl ring in close proximity to those of colchicine or
podophyllotoxin. As previously described, the ketones might
hydrogen bond with the carbonyl oxygen atom to a NH group close
to the position occupied by the carbonyl oxygen atom of podophyl-
lotoxin. Ketones with a 2,3,4-trimethoxyphenyl ring, probably due
to steric hindrance, showed a displaced carbonyl oxygen, thus sug-
gesting an explanation for their lower potency in the ketone series.
The olefins docked in similar dispositions as the corresponding
ketones (Fig. 3A). For the olefins, the displacement of the bridge
does not result in a hydrogen bond loss and is better tolerated
(Fig. 3B), as experimentally seen. In the prediction of a pharmaco-
phore for colchicine site inhibitors, the carbonyl oxygen of phenst-
Comparison of the IC50 values for all the ketone/1,1-diaryleth-
ene pairs with identical aromatic rings (i.e., 4a–p vs 5a–p) reveals
that when one compound of a pair behaves somehow in TPI assays,
the other usually follows. These results show that the carbonyl
oxygen of phenstatins is not essential, and suggest that geometri-
cal factors are possibly more important than the hydrogen bonding
acceptor capability for the higher binding affinity of phenstatins
than combretastatins. The biggest differences observed are be-
tween 4b–5b and between 4e–5e, being in both cases the ketones
more potent than the olefins. When the 2,3,4- and 3,4,5-trime-
thoxyphenyl containing compounds are compared, olefins with
bicyclic systems (indole or naphthyl) behave similarly regardless
of the substitution of the trimethoxyphenyl ring. On the other
hand, olefins containing a phenylic B ring strongly prefer the
3,4,5-trimethoxyphenyl substitution (compare m with n or o with
p).16 With regards to the B aromatic system, the most potent ana-
logues posses either an indole ring or a 3-hydroxy-4-methoxy-
phenyl or a 3-amino-4-methoxyphenyl ring, in good agreement
with previous studies.17
atins has been proposed as
a
hydrogen bond acceptor
pharmacophoric point additional to those found in combretasta-
tins. These predictions agree with the results described here. The
3-X-4-methoxyphenyl rings, N-substituted-5-indolyl and the
naphthalene units superimpose onto the methylenedioxyphenyl
ring of podophyllotoxin (Fig. 3).
We have cross-docked18 alcohols 3a–l, ketones 4a–p, and
olefins 5a–p in
a
combined podophyllotoxin–colchicine site
The ethyl group of N-ethyl-5-indolyl analogues slightly pro-
trudes over the methylenedioxyphenyl ring and displaces the in-
dole moiety. This suggests that the moiety is too large and it
using the Surflex docking program19 and AutoDock.20 Visual
examination of the docked poses revealed frequent binding with

R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
6425
Scheme 2. Synthesis of diarylmethanols 3a–l. Reagents and conditions: (i) 1 equiv nBuLi, THF, ꢀ78 °C; (ii) 2 equiv nBuLi, THF, ꢀ78 °C; (iii) Mg turnings, THF, 0 °C; (iv)
aldehydes 2a–g, 0 °C–rt; (v) NaOH, EtBr, CH2Cl2, HSO ꢀ ꢁ Bu4Nþ; (vi) NaOH, MeI, CH2Cl2, HSO ꢁ Bu4Nþ; (vii) Et3N, TBDMSCl, THF; (viii) Et3N, TBDPSCl, THF.
ꢀ
4
4
resistant to the compounds. This activity profile is different from
that shown by CA-4, which displays similar potencies against HeLa,
HL-60 and A-549, with only HT-29 showing a lower sensitivity.
Olefins are more potent than the ketones, but, as indicated for
TPI, their potency profiles are similar and parallel their tubulin
inhibitory potency, suggesting tubulin as a likely target. When
compared with combretastatins with identical substitution pat-
terns, phenstatin analogues often display more potent inhibition
of tubulin polymerization and less potent cytotoxicity. Isocombre-
tastatins A also suffer this effect, but less so than the phenstatins.
2. Conclusions
Figure 3. (A) Docked models of 4h (cyan carbon atoms) and 5h (orange carbon
atoms) showing a similar disposition for isocombretastatins and phenstatins with a
3,4,5-trimethoxyphenyl ring. (B) Docked models of 4m (blue carbon atoms) and 5m
(pink carbon atoms) showing that the absence of the carbonyl allows for the 2,3,4-
trimethoxyphenyl ring to closely resemble that of podophyllotoxin. Podophyllo-
toxin (gray carbon atoms) is shown in both figures for comparison. The protein
backbone is represented as a cartoon.
Isocombretastatins A, a new family of 1,1-diarylethene ana-
logues of combretastatins and phenstatins, have been synthesized
and assayed as tubulin polymerization inhibitors and cytotoxic
agents against several human cancer cell lines. The described com-
pounds are more potent than the parent phenones, suggesting that
the carbonyl oxygen is not essential for high potency, as had previ-
ously been suggested by a pharmacophore model proposed for the
colchicine site at tubulin. The isocombretastatins A show a modest
decrease in cytotoxic potency with respect to the parent combreta-
stains, but do not suffer of potential isomerization to inactive trans
isomers. The 1,1-diarylethene analogues constitute thus a new
family of colchicine site ligands with improved characteristics.
explains the observed modest potency decrease. Furthermore, for
5e, this steric hindrance combines with that of the the 2,3,4-trime-
thoxyphenyl, displacing further the ligand, thus explaining the ob-
served potency loss.
The synthesized compounds were assayed for their capacity to
inhibit cancer cells proliferation by the XTT method using HL-60
human leukemia, A-549 human lung carcinoma, HeLa human cer-
vix epitheloid carcinoma, and HT-29 human colon adenocarci-
noma. The results were compared with those of CA-4 and
doxorubicin (Table 1). Olefins 5d, 5n and 5p displayed nanomolar
potencies against several cell lines, and other ketones and olefins
showed low-tenths nanomolar potencies. HeLa was the most
sensitive cell line whereas HL-60, A-549 and HT-29 were more
3. Experimental
3.1. Chemistry. Materials and methods
Reagents were used as purchased without further purification.
Solvents (THF, hexanes) were dried and freshly distilled before
use according to procedures reported in the literature. Chromato-
graphic separations were performed on silica gel columns by flash

6426
R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
Table 1
Tubulin polymerization inhibitory activity and cytotoxicity IC50’s (micromolar,
lM) for 3a–f, phenstatins 4a–p and isocombretastatins 5a–p
Compound
%TPI
TPI
Cytotoxicity IC50 (lM)
Y
X
Ar1
Ar2
Ar1
X, Y
Ar2
(concn,
99 (20)
l
M)
IC50
(
l
M)
HL-60
A-549
HeLa
HT-29
CA47,9,11
Doxorubicin
3a11b,9h
3b11b,9h
3c
—
—
—
—
—
—
39,11 [1–4]
0.002
0.027
—
—
—
0.003
0.29
>10
—
>10
—
>10
>10
>10
0.25
0.5
0.12
>10
0.14
>10
3.10
>10
0.29
0.057
—
0.003
0.19
>10
—
2.5
0.032
0.24
>10
—
—
—
>10
>10
>10
2-Naphthyl
2-Naphthyl
H, OH
H, OH
H, OH
H, OH
H, OH
H, OH
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–O–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
3,4,5-TM
3,4,5-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3,4,5-TM
2,3,4-TM
3, 4, 5-TM
2,3,4-TM
3,4,5-TM
0 (20)
0 (40)
9 (20)
86 (20)
0 (20)
41 (20)
4 (20)
100 (40)
40 (20)
98 (40)
47 (20)
95 (20)
7 (20)
—
—
—
18
—
—
—
1.1
—
7.9
5.6
5.7
—
—
—
2.5 [0.4]
1.5 [0.3]
—
N-Methyl-5-indolyl
N-Methyl-5-indolyl
N-Ethyl-5-indolyl
N-Ethyl-5-indolyl
2-Naphthyl
3d11a
3e
—
—
>10
<1.0
3.3
—
—
—
<1.0
<1.0
1.7
0.044
0.03
0.035
0.45
0.016
1.8
0.037
2.5
0.03
0.03
0.2
3f11a
4a11b,9h
4b11b,9h
4c
2-Naphthyl
—
0.03
—
> 1.0
0.034
3.1
0.29
2.9
1.80 [0.56]
0.032 [0.033]
>10
0.25
—
0.008
>1.0
0.035
3.2
0.31
2.90
0.06
0.31
0.44
0.33
0.041
N-Methyl-5-indolyl
N-Methyl-5-indolyl
N-Ethyl-5-indolyl
N-Ethyl-5-indolyl
4-MeOPh
4d11a
4e
>1.0
—
4f17
4g
2.00
0.27
1.8
0.031
0.03
—
4h9a,9b,9d
4l9e
4-MeOPh
20 (20)
12 (20)
94 (20)
81 (5)
3-NO2-4-MeOPh
3-OH-4-MeOPh
3-NH2-4-MeOPh
2-Naphthyl
4n9b
4p9e
5ª
11 (20)
82 (20)
97 (20)
96 (20)
34 (20)
83 (20)
16 (20)
52 (20)
12 (20)
61 (20)
86 (20)
86 (20)
32 (20)
86 (20)
5b15
5c
2-Naphthyl
15
0.91
—
0.36
0.6
0.035
>10
0.3
N-Methyl-5-indolyl
N-Methyl-5-indolyl
N-Ethyl-5-indolyl
N-Ethyl-5-indolyl
4-MeOPh
4.7
0.7
—
6
—
25.7
—
12.2
12.7
0.8
—
0.24
0.03
>1.0
0.017
0.3
0.29
0.032
0.30
0.035
5d
5e
5f
—
0.48
0.3
2.60
0.032
0.24
0.0028
0.23
0.029
5g
>10
2.0
>10
0.026
0.27
0.028
2.70
0.031
5h15
5k
4-MeOPh
3-NO2-4-MeOPh
3-NO2-4-MeOPh
3-OH-4-MeOPh
3-OH-4-MeOPh
3-NH2-4-MeOPh
3-NH2-4-MeOPh
5l15
5m
5n15
5o
0.0029
0.06
0.0032
5p15
2.2
Selected literature values are given under brackets. TM = trimethoxyphenyl ring.
(Kieselgel 40, 0.040–0.063; Merck) or gravity column (Kieselgel 60,
0.063–0.200 mm; Merck) chromatography. TLC was performed on
precoated silica gel polyester plates (0.25 mm thickness) with UV
254 fluorescent indicator (Polychrom SI F254). 1H NMR and 13C
NMR spectra were recorded on a Bruker AC 200-SY spectrometer
at 200/50 MHz or on a Bruker SY spectrometer at 400/100 MHz.
Chemical shifts (d) are given in ppm downfield from tetramethyl-
silane as internal standard, and coupling constants (J values) are
in Hertz. GC–MS analyses were carried out on a Hewlett–Packard
another 30 min. A saturated NH4Cl solution was slowly added at
0 °C, and the mixture was extracted with CH2Cl2. The combined or-
ganic layers were dried over anhydrous Na2SO4, filtered, and con-
centrated in vacuum. The crudes were used without purification.
3.1.2. Synthesis of the benzhydrol derivatives starting from
1,2,3-trimethoxybenzene (Method B)
1,2,3-Trimethoxybenzene (3.0 M in dry THF) were reacted with
0.3 mol/mol of nBuLi (1.6 M in hexanes) at 0 °C. The reaction was
stirred for 1 h and then, 1 mol of the aldehydes in THF (100 mL/
mmol of aldehyde) per mol of nBuLi were added. The reaction mix-
ture was warmed up to room temperature and stirring was contin-
ued overnight. A saturated NH4Cl solution was slowly added and
the mixture was extracted with CH2Cl2. The combined organic lay-
ers were dried over anhydrous Na2SO4, filtered, and concentrated
in vacuum. Unreacted 1,2,3-trimethoxybenzene was removed by
column chromatography, using hexanes as eluent and the crudes
were used without further purification.
5890 Series II apparatus (70 eV). For FABHRMS analyses,
a
VG-TS250 apparatus (70 eV) was used. A Helios- UV-320 from
a
Thermo-Spectronic was used for UV experiments and absorption
spectra. HPLC analysis were run on an HP-1100 device from Agilent
Technologies or a Delta 600 device from Waters instruments, using
X-TerraÒ MS C18 5
l
m (4.6 ꢂ 150 mm), X-TerraÒ MS C8 5
lm
(4.6 ꢂ 150 mm), and X-TerraÒ MS Phenyl 5
m (4.6 ꢂ 150 mm)
l
columns with water–acetonitrile or water–methanol gradients.
Every final compound was analyzed on at least three different
column-solvent system combinations.
3.1.3. Synthesis of the benzhydrol derivatives starting from 2-
bromonaphthalene (Method C)
3.1.1. Synthesis of the benzhydrol derivatives starting from
3,4,5-trimethoxyphenyl bromide (Method A)
2-bromonaphthalene (1.0 M in dry THF) was reacted with
1 mol/mol of nBuLi (1.6 M in hexanes) at ꢀ78 °C. The reaction
was stirred for 1 h and then, 1 mol of the corresponding aldehydes
in THF (100 mL/mmol of aldehyde) per mol of nBuLi were added.
The reaction mixture was warmed up to room temperature and
stirring was continued overnight. A saturated NH4Cl solution was
slowly added and the mixture was extracted with CH2Cl2. The
3,4,5-Trimethoxyphenylmagnesium bromide (1.0 M) prepared
from 3,4,5-trimethoxyphenyl bromide and magnesium turnings
(activated with iodine) in anhydrous tetrahydrofuran was slowly
added to the corresponding aldehydes in tetrahydrofuran
(100 mL/mmol of aldehyde) at 0 °C. The reaction mixture was
warmed up to room temperature, and stirring was continued for

R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
6427
combined organic layers were dried over anhydrous Na2SO4, fil-
tered, and concentrated in vacuum. The crudes were used without
purification.
Na2SO4, filtered, and concentrated in vacuum. The residues were
purified by flash chromatography.
3.1.10. Synthesis of diarylmethanols 3
3.1.10.1. (1-Methyl-1H-indol-5-yl)(2,3,4-trimethoxyphenyl)meth-
a- nol (3c). Following general method D, 1e (500 mg, 2.4 mmol) in
dry THF (20 mL), 1.6 M nBuLi in hexanes (3.3 mL, 5.2 mmol) and
2,3,4-trimethoxybenzaldehyde (564 mg, 2.9 mmol) were reacted
to obtain alcohol 3c (550 mg, 71%), purified by flash chromatogra-
phy using hexanes/ethyl acetate 7:3 as eluent. 1H NMR d 3.65
(3H, s, 20-OCH3), 3.76 (3H, s, N-CH3), 3.85 (3H, s, 30- or 40-OCH3),
3.87 (3H, s, 30- or 40-OCH3), 6.11 (1H, br s, CH–OH), 6.46 (1H, d,
3.1.4. Synthesis of the benzhydrol derivatives starting from N-
substituted-5-bromo-1H-indoles (Method D)
The 5-bromoindoles (1.0 M in dry THF) were reacted with
2 mol/mol of nBuLi (1.6 M in hexanes) at ꢀ78 °C. The reaction
was stirred for 1 h and then a THF solution of 1 mol of aldehyde
per mol of the bromoindole (100 mL/mmol of aldehyde) was
added. The reaction mixture was warmed up to room temperature
and stirring was continued overnight. A saturated NH4Cl solution
was slowly added and the mixture was extracted with CH2Cl2.
The combined organic layers were dried over anhydrous Na2SO4,
filtered, and concentrated in vacuum. The crudes were used with-
out purification.
0
J = 3.3, H3), 6.65 (1H, d, J = 8.4, H5 ), 7.04 (1H, d, J = 3.3, H2), 7.07
(1H, d, J = 8.4, H6 ), 7.27 (2H, m, H6, H7), 7.64 (1H, br s, H4). 13C
0
NMR d 32.9 (CH3), 56.1 (CH3), 60.9 (ꢂ2) (CH3), 72.5 (CH), 101.2
(CH), 107.1 (CH), 109.1 (CH), 118.9 (CH), 120.9 (CH), 122.2 (CH),
128.4 (C), 129.3 (CH), 131.0 (C), 135.3 (C), 136.2 (C), 142.2 (C),
151.3 (C), 153.1 (C). IR (cmꢀ1) 1513, 1600, 3425.
3.1.5. Oxidations with pyridinium dichromate (Method E)
Pyridinium dichromate (PDC, 1.5 moles per mol of benzhydrol)
was added at 0 °C to a 1.0 M stirred solution of benzhydrol and 4 Å
molecular sieves in dichloromethane. After 24 h, the reaction mix-
tures were filtered through pads of Celite. The filtrates were con-
centrated in vacuum, and the residues were purified by flash
chromatography to obtain the desired substituted p-
methoxybenzophenones.
3.1.10.2. (1-Ethyl-1H-indol-5-yl)(2,3,4-trimethoxyphenyl)meth-
anol (3e). 11 mL (17.6 mmol) of 1.6 M nBuLi in hexanes was re-
acted with 1.8 g (8 mmol) of 1f in 40 mL of dry THF and with
1.88 g (9.6 mmol) of 2,3,4-trimethoxybenzaldehyde dissolved in
10 mL of dry THF to yield, after flash chromatography using hex-
ane/ethyl acetate 6:4 as eluent, 1.4 g (51%) of 3e. 1H NMR d 1.46
(3H, t, J = 7.0, CH3), 3.68 (3H, s, 20-OCH3), 3.86 (3H, s, 30- or 40-
OCH3), 3.87 (3H, s, 30- or 40-OCH3), 4.12 (2H, q, J = 7.0, N-CH2),
6.11 (1H, br s, CH–OH), 6.11 (1H, d, J = 3.3, H3), 6.66 (1H, d,
3.1.6. Oxidations with potassium permanganate (Method F)
A 0.2 M solution of the diarylmethanol in CH2Cl2 (40–50 mL)
was reacted with KMnO4 (1 mmol per mmol of alcohol) in the
0
0
J = 8.5, H5 ), 7.04 (1H, d, J = 3.3, H2), 7.08 (1H, d, J = 8.5, H6 ), 7.23
(1H, dd, J = 8.3; 1.3, H6), 7.32 (1H, d, J = 8.3, H7), 7.62 (1H, br s,
H4). 13C NMR d 15.5 (CH3), 41.1 (CH2), 56.0 (CH3), 60.8 (ꢂ2)
(CH3), 72.6 (CH), 101.3 (CH), 107.1 (CH), 109.2 (CH), 119.0 (CH),
120.7 (CH), 122.2 (CH), 127.4 (CH), 128.6 (C), 130.9 (C), 135.1
(ꢂ2) (C), 142.2 (C), 151.3 (C), 153.1 (C). IR (KBr): 1599,
3458 cmꢀ1. HRMS m/z calcd for C20H23NO4 (+Na): 364.1519, found
364.1515.
presence of n-Bu4NþHSO (about 1% w/w). The reaction mixture
ꢀ
4
was stirred for 8–12 h at room temperature and then filtered
through silica gel, using dichloromethane and ethyl acetate as elu-
ents. The organic solvent was then evaporated.
3.1.7. Wittig reactions. Synthesis of the olefin derivatives
(Method G)
The methyltriphenylphosphonium iodide (0.1 M) was sus-
pended in dry THF and cooled to ꢀ78 °C under Ar. N-butyl lithium
(1.6 M in hexanes, 0.75 moles per mol of phosphonium salt) was
added dropwise, and the resulting yellow solution was stirred for
1 h. Then a 0.1 M solution of the benzophenone (0.25 moles per
mol of phosphonium salt) in THF was added and warmed to room
temperature. Once completed the reaction mixtures were cooled to
0 °C, stopped with saturated NH4Cl solution and extracted with
dichloromethane. The organic layers were washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated in vac-
uum. The residue was purified by flash chromatography to obtain
the desired olefins.
3.1.10.3. (4-Methoxyphenyl)(2,3,4-trimethoxyphenyl)methanol
(3g). Following general method B, 7.44 mL (11.9 mmol) of 1.6 M
nBuLi in hexanes was reacted with 2.0 g (11.9 mmol) of 1b in
10 mL of dry THF and with 1.62 g (11.9 mmol) of p-methoxybenz-
aldehyde dissolved in 10 mL of dry THF to yield 3.62 g, that were
used without further purification. 1H NMR d 3.67 (3H, s, 2-OCH3),
3.80 (3H, s, 40-OCH3), 3.85 (6H, s, 3,4-OCH3), 5.89 (1H, d, J = 5,
0
0
CH–OH), 6.64 (1H, d, J = 8.6, H5), 6.86 (2H, d, J = 8.6, H3 , H5 ), 6.97
(1H, d, J = 8.6, H6), 7.28 (2H, d, J = 8.6, H2 , H6 ). 13C NMR d 55.2
(CH3), 56.0 (CH3), 60.4 (CH3), 60.7 (CH3), 71.6 (CH), 107.0 (CH),
113.6 (ꢂ2) (CH), 122.0 (CH), 127.7 (ꢂ2) (CH), 130.2 (C), 136.4 (C),
142.1 (C), 151.1 (C), 153.2 (C), 158.7 (C). IR (film): 1096, 1248,
0
0
1602, 3472 cmꢀ1
.
3.1.8. Protection of the indole nitrogen (Method H)
2 mmol of finely ground NaOH per mmol of indole derivative
and a 10% w/w (with respect to indole derivative) of tetrabutylam-
monium hydrogenosulfate were added to a 0.25 M solution of the
5-bromo-1H-indole in 50 mL of dry CH2Cl2. After stirring for 1 h at
room temperature under an Argon atmosphere, 2–3 mmol of the
alkylating agent (MeI or EtBr) was added, and the reaction was re-
fluxed until completion (as determined by TLC) for 48–144 h. The
reaction mixture was then washed with brine and the organic lay-
ers were dried, filtered, and rotary-evaporated.
3.1.10.4. (3-tertButyldimethylsiloxy-4-methoxyphenyl)(2,3,4-tri-
methoxyphenyl)methanol (3i). Following general method B,
18.75 mL (30.0 mmol) of 1.6 M nBuLi in hexanes was reacted with
5.5 g (32.7 mmol) of 1b in 20 mL of dry THF and with 2.88 g
(10.1 mmol) of 2e dissolved in 20 mL of dry THF to yield 7.54 g.
The reaction was flash chromatographied using hexanes/ethyl ace-
tate 9:1 to yield 3.22 g (69%) of 3i. 1H NMR d 0.12 (6H, s, SiCH3),
0.97 (9H, s, tertBu), 3.65 (3H, s, 2-OCH3), 3.78 (3H, s, OCH3), 3.85
(3H, s, OCH3), 3.87 (3H, s, OCH3), 5.82 (1H, d, J = 6.2, CH–OH),
0
6.64 (1H, d, J = 8.6, H5), 6.79 (1H, d, J = 8.6, H5 ), 6.86 (1H, dd,
3.1.9. Deprotection of siloxy groups (Method I)
0
0
0
J = 8.6; 2.2, H6 ), 6.88 (1H, d, J = 2.2, H2 ), 6.94 (1H, d, J = 8.6, H6 ).
13C NMR d ꢀ4.6 (ꢂ2) (CH3), 18.5 (C), 25,8 (ꢂ3) (CH3), 55.5 (CH3),
56.0 (CH3), 60.7 (ꢂ2) (CH3), 71.7 (CH), 106.9 (CH), 111.7 (CH),
119.5 (CH), 119.7 (CH), 122.1 (CH), 129.2 (C), 130.3 (C), 137.0 (C),
144.7 (C), 150.1 (C), 151.5 (C), 153.2 (C).
To a stirred 0.05 M solution of the protected phenol in dry THF
was added TBAF (1.0 M in THF, 3 moles per mol) and stirred for 1 h
under Ar. The reaction mixture was poured into water and ex-
tracted with ether, washed with brine, dried over anhydrous

6428
R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
3.1.10.5. (3-tertButyldiphenylsiloxy-4-methoxyphenyl)(3,4,5-tri-
methoxyphenyl)methanol (3j). Following method A, 2.0 g
(8.1 mmol) of 1c was added to a suspension of 304 mg (12.15 mmol)
of magnesium and 5 mg of iodine in 2.5 mL of dry THF and the
resulting white suspension was added onto a solution of 1.0 g
(2.56 mmol) of 2f dissolved in 3 mL of dry THF to yield, after 1 h
at 0 °C, 2.93 g of crude, which was purified by flash chromatography
using ethyl acetate/hexanes 1:1 as eluent to yield 1.34 g (94%) of 3j.
1H NMR d 1.09 (9H, s, tertBu), 3.60 (3H, s, 40-OCH3), 3.74 (6H, s, 3,5-
OCH3), 3.82 (3H, s, 4-OCH3), 5.49 (1H, d, J = 3.6, CH–OH), 6.41 (2H, s,
general procedure F, to yield, after 8 h, 3.0 g, that were flash chro-
matographied to yield 604 mg of 4g (17%). Mp 101–103 °C (Hex/
CH2Cl2). 1H NMR d 3.65 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.77
0
0
(6H, s, OCH3), 6.60 (1H, d, J = 8.6, H5), 6.79 (2H, d, J = 9.0, H3 , H5 ),
6.94 (1H, d, J = 8.6, H6), 7.67 (2H, d, J = 9.0, H2 , H6 ). 13C NMR d
55.4 (CH3), 56.0 (CH3), 60.8 (CH3), 61.7 (CH3), 106.8 (CH), 113.4
(ꢂ2) (CH), 124.5 (CH), 126.7 (C), 130.9 (C), 132.2 (ꢂ2) (CH), 142.0
(C), 152.2 (C), 155.7 (C), 163.4 (C), 193.9 (C). IR (KBr): 1025,
0
0
1596, 1651 cmꢀ1
.
0
0
H2, H6), 6.66 (1H, d, J = 2.2, H2 ), 6.74 (1H, d, J = 8.2, H5 ), 6.85 (1H, m,
0
3.1.11.4. (4-Methoxy-3-nitrophenyl)(2,3,4-trimethoxyphenyl)meth-
anone (4k). 567 mg (1.62 mmol) of 3k was oxidized with 1.28 g
(8.1 mmol) of KMnO4 and 20 mg of Bu4NHSO4, according to the
general procedure F, to yield, after 8 h, 220 mg (39%) that were
used without further purification. 1H NMR d 3.71 (3H, s, OCH3),
3.84 (3H, s, OCH3), 3.90 (3H, s, OCH3), 4.00 (3H, s, OCH3), 6.72
(1H, d, J = 8.6, H5), 7.10 (1H, d, J = 8.6, H6), 7.12 (1H, d, J = 8.6,
H6 ), 7.30 (6H, m, Ph), 7.66 (4H, m, Ph). 13C NMR d 14.2 (C), 26.7 (ꢂ3)
(CH3), 55.4 (CH3), 55.8 (ꢂ2) (CH3), 60.8 (CH3), 75.5 (CH), 103.2 (ꢂ2)
(CH), 118.8 (CH), 119.8 (CH), 122.1 (CH), 127.5 (4ꢂ) (CH), 129.7 (ꢂ2)
(CH), 133.5 (ꢂ2) (C), 135.4 (4ꢂ) (CH), 136.6 (C), 140.0 (C), 144.9 (C),
150.0 (C), 152.9 (ꢂ2) (C), 153.4 (C).
3.1.10.6. (4-Methoxy-3-nitrophenyl)(2,3,4-trimethoxyphenyl)
methanol (3k). Following general method B, 7.2 mL (11.5 mmol)
of 1.6 M nBuLi in hexanes was reacted with 2.0 g (11.9 mmol) of
1b in 10 mL of dry THF and with 2.08 g (11.5 mmol) of 4-meth-
oxy-3-nitro-benzaldehyde (2g) dissolved in 10 mL of dry THF to
yield 2.53 g. The reaction was flash chromatographied using hex-
anes/ethyl acetate 9:1 to yield 1.27 g (32%) of 3k. 1H NMR d 3.73
(3H, s, 2-OCH3), 3.85 (3H, s, 3- or 4-OCH3), 3.87 (3H, s, 3- or 4-
OCH3), 3.95 (3H, s, 40-OCH3), 5.88 (1H, d, J = 6.2, CH–OH), 6.65
(1H, d, J = 8.4, H5), 6.93 (1H, d, J = 8.4, H6), 7.04 (1H, d, J = 8.2,
H5 ), 8.00 (1H, dd, J = 8.6; 2.2, H6 ), 8.21 (1H, d, J = 2.2, H2 ). 13C
NMR d 56.2 (CH3), 56.9 (CH3), 61.1 (CH3), 61.9 (CH3), 107.2 (CH),
113.1 (CH), 125.2 (CH), 127.6 (CH), 130.6 (C), 135.6 (CH), 139.0
(C), 142.0 (C), 151.0 (C), 152.5 (C), 156.0 (C), 156.8 (C), 192.2 (C).
0
0
0
3.1.12. Synthesis of olefins 5
3.1.12.1. 2-[1-(2,3,4-Trimethoxyphenyl)vinyl]naphthalene (5a).
60 mg of 5a (75% isolated yield after flash chromatography using
hexanes/EtOAc 95:5) were obtained by method G from 80 mg
(0.25 mmol) of 4a with 439 mg (2.2 mmol) of CH3P+Ph3ꢁIꢀ and
1.1 mL (1.7 mmol) of nBuLi (1.6 M in hexanes) in 15 mL of dry
H5 ), 7.55 (1H, dd, J = 8.2; 2.2, H6 ), 7.87 (1H, d, J = 2.2, H2 ). 13C
NMR d 55.6 (CH3), 56.1 (CH3), 60.7 (CH3), 60.8 (CH3), 70.4 (CH),
107.2 (CH), 113.2 (CH), 121.8 (CH), 123.9 (CH), 129.0 (C), 132.3
(CH), 137.0 (C), 139.1 (C), 142.0 (C), 150.9 (C), 151.8 (C), 153.6 (C).
0
0
0
1
THF. H NMR d 3.55 (3H, s, 20-OCH3), 3.92 (3H, s, 30- or 40-OCH3),
3.93 (3H, s, 30- or 40-OCH3), 5.45 (1H, d, J = 1.5, CH2), 5.80 (1H, d,
0
0
J = 1.5, CH2), 6.74 (1H, d, J = 8.6, H5 ), 7.06 (1H, d, J = 8.6, H6 ),
7.44–7.61 (3H, m, H3, H6, H7), 7.75 (1H, br s, H1), 7.7–7.9 (3H, m,
H4, H5, H8). 13C NMR d 56.1 (CH3), 60.7 (CH3), 60.9 (CH3), 107.2
(CH), 116.0 (CH2), 125.1 (CH), 125.4 (CH), 125.7 (CH), 125.9 (CH),
126.1 (CH), 127.7 (ꢂ2) (CH), 128.3 (CH), 129.1 (C), 133.0 (C),
133.4 (C), 139.4 (C), 142.5 (C), 147.0 (C), 151.9 (C), 153.8 (C). IR
(film): 1594, cmꢀ1. HRMS m/z calcd for C21H20O3 (+Na) 343.1305,
found 343.1330.
3.1.11. Synthesis of diarylketones 4
3.1.11.1. (1-Methyl-1H-indol-5-yl)(2,3,4-trimethoxyphenyl) meth-
anone (4c). 2.0 g (6.2 mmol) of 3c was oxidized with 980 mg
(6.2 mmol) of KMnO4 and 20 mg of Bu4NHSO4, following general
procedure F, to yield, after 8 h, 1.8 g of 4c (89%) as a white solid.
1H NMR d 3.77 (3H, s, 20-OCH3), 3.83 (3H, s, NCH3), 3.92 (3H, s,
30- or 40-OCH3), 3.93 (3H, s, 30- or 40-OCH3), 6.55 (1H, d, J = 3.3,
0
H3), 6.73 (1H, d, J = 8.6, H5 ), 7.10 (1H, d, J = 3.3, H2), 7.11 (1H, d,
3.1.12.2. 2-[1-(3,4,5-Trimethoxyphenyl)vinyl]naphthalene (5b).
80 mg of 5b (55% isolated yield after flash chromatography using
hexanes/EtOAc 7:3) were obtained by method G from 150 mg
(0.46 mmol) of 4b with 941 mg (2.33 mmol) of CH3P+Ph3ꢁIꢀ and
1.2 mL (1.86 mmol) of nBuLi (1.6 M in hexanes) in 15 mL of dry
0
J = 8.6, H6 ), 7.35 (1H, d, J = 8.6, H7), 7.85 (1H, dd, J = 8.6; 1.6, H6),
8.10 (1H, d, J = 1.6, H4). 13C NMR d 33.0 (CH3), 56.1 (CH3), 61.0
(CH3), 61.8 (CH3), 103.1 (CH), 106.8 (CH), 108.9 (CH), 123.4 (CH),
124.4 (CH), 125.4 (CH), 127.8 (ꢂ2) (C), 130.4 (CH), 139.3 (C),
139.9 (C), 142.2 (C), 152.4 (C), 155.5 (C), 195.7 (C). Mp 104–
106 °C (CH2Cl2/Hex). IR (KBr): 1651, 1580 cmꢀ1. HRMS m/z calcd
for C19H19NO4 (+Na) 348.1206, found 348.1205.
1
THF. H NMR d 3.80 (6H, s, 30,50-OCH3), 3.90 (3H, s, 40-OCH3),
0
0
5.52 (1H, br s, CH2), 5.58 (1H, br s, CH2), 6.60 (2H, s, H2 , H6 ),
7.54–7.62 (3H, m, H3, H6, H7), 7.82 (1H, br s, H1), 7.7–7.9 (3H, m,
H4, H5, H8). 13C NMR d 56.2 (ꢂ2) (CH3), 61.0 (CH3), 105.8 (ꢂ2)
(CH), 114.6 (CH2), 126.2 (CH), 126.3 (CH), 126.5 (CH), 127.4 (CH),
127.7 (ꢂ2) (CH), 128.3 (CH), 133.1 (C), 133.4 (C), 137.3 (C), 137.9
(C), 138.7 (C), 150.2 (C), 153.0 (2) (C). Mp 91–93 °C (CH2Cl2/Hex).
3.1.11.2. (1-Ethyl-1H-indol-5-yl)(2,3,4-trimethoxyphenyl)meth-
anone (4e). 2.3 g (6.8 mmol) of 3e was oxidized with 1.1 g
(6.8 mmol) of KMnO4 and 23 mg of Bu4NHSO4, following general
procedure F, to yield, after 8 h, 2.1 g of 4e (91%). 1H NMR d 1.45
(3H, t, J = 7.3, CH3), 3.77 (3H, s, 20-OCH3), 3.90 (6H, s, 30,40-OCH3),
4.17 (2H, q, J = 7.3, CH2N), 6.53 (1H, d; J = 3.2, H3), 6.70 (1H, d,
IR (KBr): 1551, cmꢀ1
343.1305, found 343.1309.
. HRMS m/z calcd for C21H20O3 (+Na)
0
0
J = 8.8, H5 ), 7.08 (1H, d, J = 8.8, H6 ), 7.14 (1H, d, J = 3.2, H2), 7.35
(1H, d, J = 8.8, H7), 7.83 (1H, dd, J = 8.8, 1.8, H6), 8.06 (1H, d,
J = 1.8, H4). 13C NMR d 15.4 (CH3), 41.3 (CH2), 56.2 (CH3), 61.0
(CH3), 61.8 (CH3), 103.3 (CH), 106.8 (CH), 109.0 (CH), 123.3 (CH),
124.4 (CH), 125.6 (CH), 128.0 (C), 128.6 (CH), 130.1 (C), 138.4
(ꢂ2) (C), 142.2 (C), 152.4 (C), 155.4 (C), 195.2 (C). IR (KBr): 1597,
1651 cmꢀ1. HRMS m/z calcd for C20H21NO4 (+Na) 362.1363, found
362.1376.
3.1.12.3. 1-Methyl-5-[1-(2,3,4-trimethoxyphenyl)vinyl]-1H-indole
(5c). 110 mg of 5c (55% isolated yield after flash chromatography
using hexanes/EtOAc 8:2) were obtained by method G from
200 mg (0.6 mmol) of 4c with 1.25 g (3.1 mmol) of CH3P+Ph3ꢁIꢀ
and 1.5 mL (2.5 mmol) of nBuLi (1.6 M in hexanes) in 15 mL of
1
dry THF. H NMR d 3.55 (3H, s, 20-OCH3), 3.78 (3H, s, NCH3), 3.90
(3H, s, 30- or 40-OCH3), 3.92 (3H, s, 30- or 40-OCH3), 5.27 (1H, d,
J = 1.8), 5.66 (1H, d, J = 1.8), 6.45 (1H, d, J = 3.3, H3), 6.71 (1H, d,
0
0
J = 8.6, H6 ), 7.02 (1H, d, J = 8.6, H5 ), 7.03 (1H, d, J = 3.3, H2), 7.23–
7.31 (2H, m, H6, H7), 7.55 (1H, br s, H4). 13C NMR d 32.9 (CH3),
56.1 (CH3), 60.7 (CH3), 60.9 (CH3), 101.4 (CH), 106.9 (CH), 108.7
(CH), 113.6 (CH2), 119.3 (CH), 121.0 (CH), 125.4 (CH), 127.7 (C),
3.1.11.3. (4-Methoxyphenyl)(2,3,4-trimethoxyphenyl)methanone
(4g). 3.62 g (11.9 mmol) of 3g was oxidized with 3.76 g
(24.0 mmol) of KMnO4 and 20 mg of Bu4NHSO4, according to the

R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
6429
128.4 (C), 129.2 (CH), 130.2 (C), 133.6 (C), 136.5 (C), 147.7 (C),
151.9 (C), 153.4 (C). IR (film): 1493, 1595 cmꢀ1. HRMS m/z calcd
for C20H21NO3 (+Na) 346.1414, found 346.1399.
3.1.12.8. 1-[1-(4-Methoxyphenyl)vinyl]-3,4,5-trimethoxybenzene
(5h). The Wittig reaction (method G) produced 28 mg (25% iso-
lated yield) of 5h. 1H NMR d 3.81 (6H, s, 2 ꢂ OCH3), 3.83 (3H, s,
OCH3), 3.88 (3H, s, OCH3), 5.32 (1H, s, CH2), 5.37 (1H, s, CH2),
0
0
6.56 (2H, s, H2 , H6 ), 6.87 (2H, d, J = 8.2, H2, H6), 7.30 (2H, d,
J = 8.2, H3, H5). 13C NMR d 55.3 (CH3), 56.1 (ꢂ2) (CH3), 61.0 (CH3),
105.6 (ꢂ2) (CH), 112.6 (CH2), 113.5 (ꢂ2) (CH), 129.5 (ꢂ2) (CH),
133.7 (C), 137.6 (C), 149.6 (C), 152.9 (2) (C), 159.4 (C), 1 not ob-
served. HRMS m/z calcd for C18H20O4 (+Na) 323.1254, found
323.1271.
3.1.12.4. 1-Methyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole
(5d).
120 mg of 5d (53% isolated yield after flash chromatog-
raphy using hexanes/EtOAc 6:4) were obtained by method G from
288 mg (0.7 mmol) of 4d with 850 mg (2.1 mmol) of CH3P+Ph3ꢁIꢀ
and 1.1 mL (1.7 mmol) of nBuLi (1.6 M in hexanes) in 15 mL of
1
dry THF. H NMR d 3.80 (3H, s, N- or -OCH3), 3.82 (6H, s, 30,50-
OCH3), 3.93 (3H, s, N- or -OCH3), 5.41 (1H, s, CH2), 5.47 (1H, s,
3.1.12.9. 1-[1-(3-tertButyldimethylsiloxy-4-methoxyphenyl)vinyl]-
2,3,4-trimethoxybenzene (5i). 480 mg of 5i (48% isolated yield
after flash chromatography using hexanes/EtOAc 9:1) were ob-
tained by method G from 1.00 g (2.31 mmol) of 4i with 3.7 g
(9.2 mmol) of CH3P+Ph3ꢁIꢀ and 4.31 mL (6.90 mmol) of nBuLi
(1.6 M in hexanes) in 40 mL of dry THF. 1H NMR d 0.13 (6H, s,
SiCH3), 0.98 (9H, s, tertBu), 3.54 (3H, s, OCH3), 3.79 (3H, s, OCH3),
3.85 (3H, s, OCH3), 3.89 (3H, s, OCH3), 5.17 (1H, d, J = 1.4, CH2),
5.51 (1H, d, J = 1.4, CH2), 6.67 (1H, d, J = 8.6, H5), 6.73 (1H, d,
0
0
CH2), 6.50 (1H, d, J = 3.2, H3), 6.66 (2H, s, H2 , H6 ), 7.07 (1H, d,
J = 3.2, H2), 7.30 (2H, m, H6, H7), 7.64 (1H, br s, H4). 13C NMR d
33.0 (CH3), 56.2 (ꢂ2) (CH3), 60.1 (CH3), 101.4 (CH), 106.2 (ꢂ2)
(CH), 108.9 (CH), 112.5 (CH2), 120.9 (CH), 122.5 (CH), 128.4 (C),
129.4 (CH), 132.8 (C), 136.6 (C), 137.8 (C), 138.5 (C), 151.2 (C),
152.9 (ꢂ2) (C). IR (film): 1582 cmꢀ1
. HRMS m/z calcd for
C20H21NO3 (+Na) 346.1414, found 346.1430.
3.1.12.5. 1-Ethyl-5-[1-(2,3,4-trimethoxyphenyl)vinyl]-1H-indole
(5e). 60 mg of 5e (40% isolated yield after flash chromatography
using hexanes/EtOAc 8:2) were obtained by method G from
150 mg (0.4 mmol) of 4e with 888 mg (2.1 mmol) of CH3P+Ph3ꢁIꢀ
and 1.1 mL (1.7 mmol) of nBuLi (1.6 M in hexanes) in 25 mL of
0
0
J = 8.6, H6), 6.80 (1H, dd, J = 8.6; 2.2, H6 ), 6.89 (1H, d, J = 2.2, H2 ),
6.93 (1H, d, J = 8.6, H5 ). 13C NMR d ꢀ4.5 (ꢂ2) (CH3), 18.5 (C),
0
25.8 (ꢂ3) (CH3), 55.5 (CH3), 56.0 (CH3), 60.7 (CH3), 60.8 (CH3),
106.9 (CH), 111.4 (CH), 113.7 (CH2), 119.4 (CH), 120.3 (CH), 125.2
(CH), 132.2 (C), 134.9 (C), 142.3 (C), 144.6 (C), 146.4 (C), 150.6
(C), 151.6 (C), 153.5 (C).
1
dry THF. H NMR d 1.47 (3H, t, J = 7.3, CH3), 3.55 (3H, s, 20-OCH3),
3.88 (3H, s, 30- or 40-OCH3), 3.91 (3H, s, 30- or 40-OCH3), 4.16 (2H,
q, J = 7.3, CH2N), 5.24 (1H, d, J = 1.8, CH2), 5.64 (1H, d, J = 1.8,
3.1.12.10. 1-[1-(3-tertButyldiphenylsiloxy-4-methoxyphenyl)vinyl]-
3,4,5-trimethoxybenzene (5j). 177 mg of 5j (57% isolated yield
after flash chromatography using hexanes/EtOAc 9:1) were ob-
tained by method G from 310 mg (0.56 mmol) of 4j with 653 mg
(1.62 mmol) of CH3P+Ph3ꢁIꢀ and 0.84 mL (1.35 mmol) of nBuLi
(1.6 M in hexanes) in 7 mL of dry THF. 1H NMR d 1.14 (9H, s, tert-
Bu), 3.62 (3H, s, 4- or 40-OCH3), 3.74 (6H, s, 30,50-OCH3), 3.89 (3H, s,
4- or 40-OCH3), 5.10 (1H, d, J = 1.4, CH2), 5.15 (1H, d, J = 1.4, CH2),
0
CH2), 6.44 (1H, d, J = 3.1, H3), 6.70 (1H, d, J = 8.4, H5 ), 7.00 (1H, d,
0
J = 8.4, H6 ), 7.09 (1H, d, J = 3.1, H2), 7.27 (2H, m, H5, H6), 7.53
(1H, br s, H4). 13C NMR d 15.7 (CH3), 41.0 (CH2), 56.1 (CH3), 60.7
(ꢂ2) (CH3), 101.6 (CH), 107.0 (CH), 108.8 (CH), 113.5 (CH2), 119.4
(CH), 120.9 (CH), 125.4 (CH), 127.3 (CH), 128.6 (C), 130.2 (C),
133.5 (C), 135.6 (C), 147.7 (ꢂ2) (C), 151.9 (C), 153.4 (C). IR (film):
1595 cmꢀ1. HRMS m/z calcd for C21H23NO3 (+Na) 360.1570, found
360.1565.
0
6.42 (2H, s, H2, H6), 6.72 (1H, d, J = 1.8, H2 ), 6.73 (1H, d, J = 8.2,
0
0
H5 ), 6.85 (1H, dd, J = 8.2, 1.8, H6 ), 7.35 (6H, m, Ph), 7.70 (4H, m,
Ph). 13C NMR d 19.8 (C), 26.8 (ꢂ3) (CH3), 55.5 (CH3), 56.0 (ꢂ2)
(CH3), 61.0 (CH3), 105.5 (ꢂ2) (CH), 111.7 (CH), 112.4 (CH2), 120.2
(CH), 121.4 (CH), 127.5 (ꢂ4) (CH), 129.7 (ꢂ2) (CH), 133.5 (ꢂ2)
(C), 133.7 (ꢂ2) (C), 135.6 (ꢂ4) (CH), 137.5 (C), 144.7 (C), 149.5
(C), 150.5 (C), 152.7 (ꢂ2) (C).
3.1.12.6. 1-Ethyl-5-[1-(3,4,5-trimethoxyphenyl)vinyl]-1H-indole
(5f). 77 mg of 5f (52% isolated yield after flash chromatography
using hexanes/EtOAc 8:2) were obtained by method G from
150 mg (0.4 mmol) of 4f with 880 mg (2.2 mmol) of CH3P+Ph3ꢁIꢀ
and 1.1 mL (1.7 mmol) of nBuLi (1.6 M in hexanes) in 15 mL of
1
dry THF. H NMR d 1.50 (3H, t, J = 7.3, CH3), 3.82 (6H, s, 30,50-
OCH3), 3.91 (3H, s, 40-OCH3), 4.19 (2H, q, J = 7.3, CH2N), 5.37 (1H,
d, J = 1.5, CH2), 5.45 (1H, d, J = 1.5, CH2), 6.50 (1H, d, J = 3.3, H3),
3.1.12.11. 1-[1-(3-Nitro-4-methoxyphenyl)vinyl]-2,3,4-trimethoxy-
benzene (5k). 58 mg of 5k (27% isolated yield after flash chroma-
tography using hexanes/EtOAc 9:1) were obtained by method G
from 220 mg (0.56 mmol) of 4k with 1.02 g (2.54 mmol) of
CH3P+Ph3ꢁIꢀ and 1.38 mL (2.22 mmol) of nBuLi (1.6 M in hexanes)
0
0
6.62 (2H, s, H2 , H6 ), 7.14 (1H, d, J = 3.3, H2), 7.24 (1H, dd, J = 8.8;
1.4, H6), 7.32 (1H, d, J = 8.8, H7), 7.65 (1H, br s, H4). 13C NMR d
15.5 (CH3), 41.1 (CH2), 56.2 (ꢂ2) (CH3), 60.9 (CH3), 101.6 (CH),
106.2 (ꢂ2) (CH), 108.8 (CH), 112.3 (CH2), 121.0 (CH), 122.3 (CH),
127.5 (CH), 128.0 (C), 128.6 (C), 129.1 (C), 132.7 (C), 138.4 (C),
151.2 (C), 152.9 (ꢂ2) (C). IR (film): 1504, 1580 cmꢀ1. HRMS m/z
calcd for C21H23NO3 (+Na) 360.1570, found 360.1560.
1
in 7 mL of dry THF. H NMR d 3.55 (3H, s, 20-OCH3), 3.84 (3H, s,
30- or 40-OCH3), 3.89 (3H, s, 30- or 40-O CH3), 3.90 (3H, s, 4-OCH3),
5.30 (1H, s, CH2), 5.58 (1H, s, CH2), 6.68 (1H, d, J = 8.4, H5), 6.94
0
(1H, d, J = 8.4, H6), 7.00 (1H, d, J = 8.8, H5 ), 7.47 (1H, dd, J = 8.8,
2.2, H6 ), 7.78 (1H, d, J = 2.2, H2 ). 13C NMR d 56.1 (CH3), 56.6
(CH3), 60.7 (CH3), 61.0 (CH3), 107.3 (CH), 113.1 (CH), 115.9 (CH2),
123.5 (CH), 125.2 (CH), 127.7 (C), 132.1 (CH), 134.6 (C), 139.3 (C),
142.4 (C), 144.6 (C), 151.5 (C), 152.2 (C), 154.1 (C). IR (film):
0
0
3.1.12.7. 1-[1-(4-Methoxyphenyl)vinyl]-2,3,4-trimethoxybenzene
(5g). 27 mg of 5g (20% isolated yield after flash chromatography
using hexanes/EtOAc 9:1) were obtained by method G from
137 mg (0.45 mmol) of 4g with 731 mg (1.8 mmol) of CH3P+Ph3ꢁIꢀ
and 0.85 mL (1.36 mmol) of nBuLi (1.6 M in hexanes) in 7 mL of dry
THF. 1H NMR d 3.55 (3H, s, 20-OCH3), 3.80 (3H, s, 30-, 40 or 4-OCH3),
3.86 (3H, s, 30-, 40 or 4-OCH3), 3.89 (3H, s, 30-, 40 or 4-OCH3), 5.18
(1H, d, J = 1.4, CH2), 5.54 (1H, d, J = 1.4, CH2), 6.67 (1H, d, J = 8.6,
1531, 1598 cmꢀ1
.
3.1.12.12. 1-[1-(3-Nitro-4-methoxyphenyl)vinyl]-3,4,5-trime-
thoxybenzene (5l). 114 mg of compound 5l were obtained in 58%
yield by Wittig reaction (method G). 1H NMR d 3.79 (6H, s,
2 ꢂ OCH3), 3.85 (3H, s, OCH3), 3.95 (3H, s, OCH3), 5.41 (2H, s,
0
0
H5), 6.83 (2H, d, J = 8.8, H3 , H5 ), 6.95 (1H, d, J = 8.6, H6), 7.25 (2H,
d, J = 8.8, H3, H5). 13C NMR d 55.3 (CH3), 56.0 (CH3), 60.7 (CH3),
60.9 (CH3), 106.9 (CH), 113.5 (ꢂ2) (CH), 113.7 (CH2), 125.2 (CH),
127.8 (ꢂ2) (CH), 129.4 (C), 134.5 (C), 142.1 (C), 146.3 (C), 151.7
0
0
CH2), 6.48 (2H, s, H2 , H6 ), 7.04 (1H, d, J = 8.6, H5), 7.50 (1H, dd;
J = 8.6, 2.3, H6), 7.83 (1H, d, J = 2.3, H2). 13C NMR d 56.2 (ꢂ2)
(CH3), 56.6 (CH3), 60.9 (CH3), 105.5 (ꢂ2) (CH), 113.3 (CH), 114.7
(CH2), 125.1 (CH), 133.8 (CH), 136.2 (C), 139.3 (C), 142,3 (C),
(C), 153.5 (C), 159.2 (C). IR (film): 1500, 1602 cmꢀ1
.

6430
R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
147.1 (C), 147.6 (C), 152.5 (C), 153.1 (ꢂ2) (C). HRMS m/z calcd for
3.3. Tubulin assembly
C18H19NO6 (+Na): 368.1105, found 368.1124.
In vitro tubulin self-assembly was monitored turbidimetrically
at 450 nm, using a thermostated Thermo-Spectronic Helios
a spec-
3.1.12.13. 5-[1-(2,3,4-Trimethoxyphenyl)vinyl]-2-methoxyphenol
(5m). Following method I, 0.56 mL of 1.0 M TBAF solution in THF
was added to 80 mg (0.19 mmol) of 5i dissolved in 2 mL of dry
THF at room temperature under Ar atmosphere. After 1 h, the solu-
tion was poured onto water and extracted with ether. The organic
layers were washed with brine, dried over anhydrous Na2SO4 and
trophotometer fitted with a temperature controller and a circulat-
ing water carrousel system. The ligands were dissolved in DMSO
and stored at ꢀ20 °C. The amount of DMSO in the assays was 4%,
which has been reported not to interfere with the assembly pro-
cess.24 The increase in turbidity was followed simultaneously in
a batch of six cuvettes (containing 1.0 mg/mL MTP in 0.1 M MES
buffer, 1 mM EGTA, 1 mM MgCl2, 1 mM, b-ME, 1.5 mM GTP, pH
6.7, and the measured ligand concentration), with a control (i.e.,
with no ligand) always being included.
The samples were preincubated for 30 min at 20 °C in order to
allow binding of the ligand, and were cooled on ice for 10 min.
The cuvettes were then placed in the spectrophotometer at 4 °C.
The assembly process was initiated by a shift in the temperature
to 37 °C. The IC50 was calculated as the concentration of drug caus-
ing 50% inhibition of polymerization after 20 min of incubation and
was determined graphically. At least two independent experiments
(or more when required for the most potent inhibitors) with differ-
ent MTP preparations were carried out for each compound tested.
1
evaporated to yield 57 mg (95%) of 5m. H NMR d 3.57 (3H, s, 20-
OCH3), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3), 3.88 (3H, s, OCH3),
5.18 (1H, d, J = 1.6, CH2), 5.55 (1H, d, J = 1.6, CH2), 6.66 (1H, d,
0
J = 8.6, H5 ), 6.72 (1H, d, J = 8.2, H3), 6.96 (1H, br d, J = 8.2, H4),
6.92 (1H, d, J = 8.6, H6 ), 6.96 (1H, br s, H6). 13C NMR d 56.0 (CH3),
0
56.1 (CH3), 60.8 (CH3), 60.9 (CH3), 107.0 (CH), 110.1 (CH), 113.0
(CH), 114.1 (CH2), 118.7 (CH), 125.2 (CH), 129.2 (C), 135.4 (C),
142.3 (C), 145.2 (ꢂ2) (C), 146.2 (ꢂ2) (C), 153.5 (C). IR (film):
1593, 3415 cmꢀ1
.
3.1.12.14. 5-[1-(3,4,5-Trimethoxyphenyl)vinyl]-2-methoxyphenol
(5n). Following the general procedures A, E, G and I, 5n was ob-
tained in 57% yield. 1H NMR d 3.81 (6H, s, 2 ꢂ OCH3), 3.87 (3H, s,
OCH3), 3.90 (3H, s, OCH3), 5.30 (1H, d, J = 1.4, CH2), 5.37 (1H, d,
3.4. XTT procedure
0
0
J = 1.4, CH2), 6.55 (2H, s, H2 , H6 ), 6,80 (1H, d, J = 8.2, H3), 6.85
(1H, dd, J = 8.2, 1.8, H4), 6.97 (1H, d, J = 1.8, H6). 13C NMR d 56.0
(ꢂ3) (CH3), 61.0 (CH3), 105.7 (ꢂ2) (CH), 110.2 (CH), 113.0 (CH2),
114.5 (CH), 120.0 (CH), 127.7 (C), 134.7 (C), 137.5 (C), 145.2 (C),
146.5 (C), 149.6 (C), 152.8 (ꢂ2) (C). HRMS m/z calcd for C18H20O5
(+Na) 339.1203, found 339.1219.
100
l
L of exponentially growing HeLa (1.5 ꢂ 103 cells/well),
HT-29 (3 ꢂ 103 cells/well), or A-549 (5 ꢂ 103 cells/well) cells were
seeded in 96-well flat-bottomed microtiter plates, and incubated at
37 °C in a humidified atmosphere of 5% CO2/95% air for 24 h to al-
low the cells attach to the plates. HL-60 cells were seeded at
3 ꢂ 103 (100
lL) cells per well. Then, cells were incubated with dif-
3.1.12.15. 5-[1-(2,3,4-Trimethoxyphenyl)vinyl]-2-methoxyaniline
(5o). 100 mg (1.54 mmol) of Zinc turnings was added to a stirred
suspension of 57 mg (0.17 mmol) of 5k in 10 mL of CH2Cl2 and
2 mL of glacial acetic acid under Ar. After 12 h, the reaction mixture
was filtered through Celite. The filtrate was concentrated under
vacuum to obtain 52 mg (97%) of compound 5o.1H NMR d 3.56
(3H, s, 20-OCH3), 3.82 (3H, s, OCH3), 3.85 (3H, s, OCH3), 3.87 (3H,
s, OCH3), 5.15 (1H, s, CH2), 5.55 (1H, s, CH2), 6.66 (4H, m, H4, H6,
ferent concentrations of the assayed compound at 37 °C under the
5% CO2/95% air atmosphere for 72 h. Cell proliferation was quanti-
fied using the XTT (30-[1-(phenylamino)carbonyl]-3,4-tetrazolium-
bis(4-methoxy-6-nitro)benzene sulfonic acid sodium salt hydrate)
cell proliferation kit (Roche Molecular Biochemicals, Mannheim,
Germany) following the manufacturer0s instructions. Briefly, a
freshly prepared mixture solution (50 lL) of XTT labeling reagent
and PMS (N-methyldibenzopyrazine methyl sulfate) electron cou-
pling reagent was added to each well. The resulting mixtures were
further incubated for 4 h in a humidified atmosphere (37 °C, 5%
CO2), and the absorbance of the formazan product generated was
measured with a microtiter plate reader at a test wavelength of
490 nm and a reference wavelength of 655 nm. The IC50 (50%
inhibitory concentration) was then calculated as the drug concen-
tration causing a 50% inhibition of cell proliferation. Data are
shown as mean values of three independent experiments per-
formed in triplicate.
H5 , H6 ), 6.92 (1H, d, J = 8.2, H3). 13C NMR d 55.6 (CH3), 56.0
(CH3), 60.8 (CH3), 60.9 (CH3), 106.8 (CH), 109.9 (CH), 113.7 (CH2),
114.2 (CH), 118.0 (CH), 125.3 (CH), 129.5 (C), 134.8 (C), 142.3 (C),
146.5 (ꢂ2) (C), 147.5 (C), 151.7 (C), 153.4 (C). IR (film): 1596,
0
0
3367 cmꢀ1
.
3.1.12.16. 5-[1-(3,4,5-Trimethoxyphenyl)vinyl]-2-methoxyaniline
(5p). To a stirred suspension of 5l in CH2Cl2:glacial acetic acid 1:5
(0.1 M) under Ar was added 10 equiv of Zinc turnings. After 12 h,
the reaction mixture was filtered through Celite. The filtrate was
concentrated under vacuum to obtain 94 mg of compound 5p in
quantitative yield. 1H NMR d 3.82 (6H, s, 2 ꢂ OCH3), 3.87 (6H, s,
2 ꢂ OCH3), 5.27 (1H, d, J = 1.2, CH2), 5.34 (1H, d, J = 1.2, CH2), 6.56
3.5. Molecular modeling
The compounds were docked into the colchicine site of tubulin
following a described protocol.25 The X-ray structures of the tubu-
lin complexes with podophyllotoxin and DAMA-colchicine were
retrieved from the protein data bank,26 while chains C, D and E
and the corresponding hetero-groups were removed by hand.
The pdb files were energy-minimized and subjected to molecular
dynamics simulations at 300 K.27 We initially restrained the back-
bone, and then it was set free. The relaxed structures were super-
imposed and a combined site was generated by shifting the
tubulin-podophyllotoxin complex 30 Å along the X axes. The com-
bined tubulin sites and the podophyllotoxin and DAMA-colchicine
ligands were used to generate a combined protomol with the Sur-
flex docking program.19 The individual sites were also used in sep-
arate docking experiments with AutoDock 3.20 The synthesised
compounds together with roughly 300 combretastatin and phenst-
(2H, s, H2 , H6 ), 6.74 (3H, s, H3, H4, H6). 13C NMR d 55.6 (CH3),
56.2 (ꢂ2) (CH3), 61.0 (CH3), 105.7 (ꢂ2) (CH), 109.9 (CH), 112.4
(CH2), 115.3 (CH), 119.0 (CH), 134.1 (C), 135.4 (C), 137.6 (C),
137.8 (C), 147.5 (C), 150.0 (C), 152.8 (ꢂ2) (C). HRMS m/z calcd for
C18H21NO4 (+Na): 338.1363, found 338.1379.
0
0
3.2. Tubulin isolation
Calf brain microtubule protein (MTP) was purified by two cycles
of temperature-dependent assembly/disassembly, according to the
method of Shelanski,21 modified as described in the literature.22
The MTP solution was stored at ꢀ80 °C. Protein concentrations
were determined by the method of Bradford,23 using BSA as
standard.

R. Álvarez et al. / Bioorg. Med. Chem. 17 (2009) 6422–6431
6431
atin analogues were manually constructed in silico28 and docked
into the combined sites (cross-docking), in an attempt to better
reproduce the receptor flexibility by using different configurations
of the protein.18 Additionally, the test set of ACD compounds used
in the Surflex validation were equally docked. These compounds
were considered a negative control group: that is, lacking biologi-
cal activity. The combined results were analyzed by receiver oper-
ating characteristics (ROCs);29 the ACD compounds and the
analogues of the colchicine site with published TPI worse than
Álvarez, C.; Álvarez, R.; Corchete, P.; Pérez-Melero, C.; Peláez, R.; Medarde, M.
Bioorg. Med. Chem. Lett. 2007, 17, 3417; (i) Maya, A. B. S.; Pérez-Melero, C.;
Mateo, C.; Alonso, D.; Fernández, J. L.; Gajate, C.; Mollinedo, F.; Peláez, R.;
Caballero, E.; Medarde, M. J. Med. Chem. 2005, 48, 556.
10. (a) Mateo, C.; López, V.; Medarde, M.; Peláez, R. Chem. Eur. J. 2007, 13, 7246; (b)
Mateo, C.; Álvarez, R.; Pérez-Melero, C.; Peláez, R.; Medarde, M. Bioorg. Med.
Chem. Lett. 2007, 17, 6316; (c) Mateo, C.; Pérez-Melero, C.; Peláez, R.; Medarde,
M. J. Org. Chem. 2005, 70, 6544.
11. (a) Álvarez, C.; Álvarez, R.; Corchete, P.; López, J. L.; Pérez-Melero, C.; Peláez, R.;
Medarde, M. Bioorg. Med. Chem. 2008, 16, 5952; (b) Álvarez, C.; Álvarez, R.;
Corchete, P.; López, J. L.; Pérez-Melero, C.; Peláez, R.; Medarde, M. Bioorg. Med.
Chem. 2008, 16, 8999.
20 lM were considered inactive. The enrichment factors achieved
12. The term isocombretastatin A has previously been used for the regioisomer of
combretastatin. However, the
A particle is usually aplied for stilbenic
were similar to those described for similar systems. The structures
of the best scored complexes in the colchicine and podophyllotoxin
sites were inspected visually and compared with the TPI results.30
combretastatins, thus the term isocombretastatins A: Singh, S. B.; Pettit, G. R.
Synth. Commun. 1987, 17, 877.
13. (a) Cullen, M. D.; Sarkar, T.; Hamel, E.; Hartman, T. L.; Watson, K. M.; Buckheit,
R. W.; Pannecouque, C.; De Clercq, E.; Cushman, M. Bioorg. Med. Chem. Lett.
2008, 18, 469; (b) Cullen, M. D.; Cheung, Y. F.; Houslay, M. D.; Hartman, T. L.;
Watson, K. M.; Buckheit, R. W.; Pannecouque, C.; De Clercq, E.; Cushman, M.
Bioorg. Med. Chem. Lett. 2008, 18, 1530; (c) Deng, B. L.; Hartman, T. L.; Buckheit,
R. W.; Pannecouque, C.; De Clercq, E.; Cushman, M. J. Med. Chem. 2006, 49,
5316; (d) Deng, B. L.; Hartman, T. L.; Buckheit, R. W.; Pannecouque, C.; De
Clercq, E.; Fanwick, P. E.; Cushman, M. J. Med. Chem. 2005, 48, 6140.
14. (a) Wu, L.; Zhang, L. H.; Pedemonte, M.; Nazriuddin, S.; Lai, Y.; Lu, L.; Schafer,
P.; Muller, G.; Stirling, D.; Payvandi, F. Blood 2003, 102, 209B; (b) Zhang, L. H.;
Lu, L.; Wit, L.; Robarge, M.; Liu, W.; Chen, R. S.; Muller, G. W.; Stirling, D.;
Payvandi, F. Clin. Cancer Res. 2003, 9, 6142S; (c) Zhang, L. H.; Wu, L.; Raymon,
H. K.; Chen, R. S.; Corral, L.; Shirley, M. A.; Narla, R. K.; Gamez, J.; Muller, G.
W.; Stirling, D. I.; Bartlett, J. B.; Schafer, P. H.; Payvandi, F. Cancer Res. 2006,
66, 951.
15. (a) Álvarez, C. Doctoral Thesis, University of Salamanca, 2006.; (b) Alami, M.;
Brion, J. D.; Provot, O.; Peyrat, J. F.; Messaoudi, S.; Hamze, A.; Giraud, A.; Bignon,
J.; Bakala, J.; Liu, J.-M. Preparation of Iso CA-4 and analogs as potent cytotoxic
agents and inhibitors of polymerization of tubulin WO 2008122620, 2008, p 78.
16. When both substitution patterns lead to inactive or low potency compounds,
as, for instance, in g and h, such a comparison is not possible.
17. (a) Liou, J.-P.; Wu, C.-Y.; Hsieh, H.-P.; Chang, C.-Y.; Chen, C.-M.; Kuo, C.-C.;
Chang, J.-Y. J. Med. Chem. 2007, 50, 4548; (b) Hu, L.; Jiang, J.-D.; Qu, J.; Li, Y.; Jin,
J.; Li, Z.-R.; Boykin, D. W. Bioorg. Med. Chem. Lett. 2007, 17, 3613; (c) Ref. 11a.
18. (a) Sotriffer, C. A.; Dramburg, I. J. Med. Chem. 2005, 48, 3122; (b) Ragno, R.;
Frasca, S.; Manetti, F.; Brizzi, A.; Massa, S. J. Med. Chem. 2005, 48, 200; (c)
Zentgraf, M.; Fokkens, J.; Sotriffer, C. A. ChemMedChem 2006, 1, 1355.
19. version 2.1 was used: (a) Jain, A. N. J. Med. Chem. 2003, 46, 499; (b) Jain, A. N.
Surflex-Dock 2.1: J. Comput. Aided Mol. Des. 2007, 21, 281.
20. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
21. Shelanski, M. L.; Gaskin, F.; Cantor, C. R. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 765.
22. Dumortier, C.; Gorbunoff, M.; Andreu, J. M.; Engelborghs, Y. Biochemistry 1996,
35, 4387.
23. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
24. Han, Y.; Chaudhary, A. G.; Chordia, M. D.; Sackett, D. L.; Pérez-Ramirez, B.;
Kingston, D. G.; Bane, S. Biochemistry 1996, 35, 14173.
25. Peláez, R.; López, J. L.; Medarde, M. Application of Chemoinformatic Tools for
The Analysis of Virtual Screening Studies of Tubulin Inhibitors. In Advances in
Soft Computing; Corchado, E., Corchado, J. M., Abraham, A., Eds.; Springer:
Berlin, 2007; Vol. 44, pp 411–417.
26. http://www.wwpdb.org/.
27. Discover program from http://www.accelrys.com/ was used.
28. http://www.chemaxon.com/.
Acknowledgments
We thank the Consejería de Educación de la Junta de Castilla y
León (Programa de Apoyo a Proyectos de Investigación, EDU/940/
2009, SA067A09), Spanish MEC (Ref CTQ2004-00369/BQU), the
EU (Structural Funds) and the Ministerio de Ciencia e Innovación
of Spain (SAF2008-04242, SAF2008-02251, and RD06/0020/
1037—Red Temática de Investigación Cooperativa en Cáncer, Insti-
tuto de Salud Carlos III) for financial support. C. A. and R. A. thank
the Spanish MEC for FPI predoctoral grants and R. A. thanks the
Fundación Ramón Areces for a predoctoral grant.
References and notes
1. Microtubules: Hyams, J., Lloyd, C. W., Eds.; Wiley-Liss: New York, 1994.
2. Honore, S.; Pasquier, E.; Braguer, D. Cell. Mol. Life Sci. 2005, 62, 3039.
3. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer 2004, 4, 253.
4. Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.; Sobel, A.;
Knossow, M. Nature 2004, 428, 198.
5. (a) Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Med. Res. Rev. 2008, 28,
155; (b) Zefirova, O. N.; Diikov, A. G.; Zyk, N. V.; Zefirov, N. S. Russ. Chem. Bull.
2007, 56, 680; (c) Nguyen, T. L.; McGrath, C.; Hermone, A. R.; Burnett, J. C.;
Zaharevitz, D. W.; Day, B. W.; Wipf, P.; Hamel, E.; Gussio, R. J. Med. Chem. 2005,
48, 6107; (d) Ducki, S.; Mackenzie, G.; Lawrence, N. J.; Snyder, J. P. J. Med. Chem.
2005, 48, 457; (e) Brown, M. L.; Rieger, J. M.; Macdonald, T. L. Bioorg. Med. Chem.
2000, 8, 1433; (f) terHaar, E.; Rosenkranz, H. S.; Hamel, E.; Day, B. W. Bioorg.
Med. Chem. 1996, 4, 1659.
6. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989, 28, 6984.
7. Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. J. Med.
Chem. 2006, 49, 3033.
8. Lippert, J. W. Bioorg. Med. Chem. 2007, 15, 605.
9. (a) Cushman, M.; Nagarathnam, D.; Gopal, D.; He, H. M.; Lin, C. M.; Hamel, E. J.
Med. Chem. 1992, 35, 2293; (b) Pettit, G. R.; Toki, B.; Herald, D. L.; Verdier-
Pinard, P.; Boyd, M. R.; Hamel, E.; Pettit, R. K. J. Med. Chem. 1998, 41, 1688; (c)
Pettit, G. R.; Grealish, M. P.; Herald, D. L.; Boyd, M. R.; Hamel, E.; Pettit, R. K. J.
Med. Chem. 2000, 43, 2731; (d) Liou, J. P.; Chang, C. W.; Song, J. S.; Yang, Y. N.;
Yeh, C. F.; Tseng, H. Y.; Lo, Y. K.; Chang, Y. L.; Chang, C. M.; Hsieh, H. P. J. Med.
Chem. 2002, 45, 2556; (e) Liou, J. P.; Chang, J. Y.; Chang, C. W.; Chang, C. Y.;
Mahindroo, N.; Kuo, F. M.; Hsieh, H. P. J. Med. Chem. 2004, 47, 2897; (f) Liou, J.
P.; Chang, Y. L.; Kuo, F. M.; Chang, C. W.; Tseng, H. Y.; Wang, C. C.; Yang, Y. N.;
Chang, J. Y.; Lee, S. J.; Hsieh, H. P. J. Med. Chem. 2004, 47, 4247; (g) Duan, J. X.;
Cai, X.; Meng, F.; Lan, L.; Hart, C.; Matteucci, M. J. Med. Chem. 2007, 50, 1001; (h)
29. Triballeau, N.; Acher, F.; Brabet, I.; Pin, J. P.; Bertrand, H. O. J. Med. Chem. 2005,
48, 2534.
30. Jmol: an open-source Java viewer for chemical structures in 3D. http://
www.jmol.org/.